locusts
Basil el Jundi1 and Uwe Homberg1
1
Department of Biology, Animal Physiology, University of Marburg, 35032 Marburg, Germany
el Jundi B, Homberg U. Many migrating insects rely on the plane of sky polarization as a cue to detect spatial directions. In desert locusts (Schistocerca gregaria), as in other insects, polarized light is perceived by photoreceptors in a specialized dorsal eye region. Desert locusts occur in two phases, a gregarious swarming phase that migrates during the day and a solitarious, nocturnal phase. Neurons in a small brain area, the anterior optic tubercle (AOTu), are critically involved in processing polarized light in the locust brain. While polarization-sensitive (POL) intertubercle cells, LoTu1 and TuTu1, interconnect the AOTu of both hemispheres, TuLAL1 neurons transmit sky compass signals to a polarization compass in the central brain. To better understand possible adaptations of the polarization vision system to a diurnal vs. nocturnal life style we analyzed receptive field properties, intensity/response relationships and daytime dependence of responses of the AOTu neurons in gregarious and solitarious locusts. Surprisingly, no differences in the physiology of these neurons were found between the two locust phases suggesting that both phases rely on the same sky navigation system. Instead, clear differences were observed between different types of AOTu neurons.
While TuTu1 and TuLAL1 neurons encoded E-vector orientation independent of light intensity and would thus be operational in bright daylight, LoTu1 was inhibited by high light intensity and provided strong polarization signaling only at dim light conditions.
The presence of a high and low intensity polarization channel might, therefore, allow both phases to use the same polarization coding system despite their different activity cycles.
I N T R O D U C T I O N
Many navigating animals rely on external visual signals for spatial orientation. Insects use mainly two mechanisms to calculate moving directions during flight or walking. In familiar areas they are able to use visual landmarks as directional cues while in unknown terrains and during long-distance migrations, compass signals from the sky are more relevant (Giurfa and Capaldi 1999; Collett and Collett 2000). Besides the direct position of the sun, the plane of sky polarization serves as a crucial reference for spatial directions during seasonal migration or homing
Correspondence to: Uwe Homberg, Fachbereich Biologie, Tierphysiologie, Universität Marburg, D-35032 Marburg, Germany (E-mail: homberg@biologie.uni-marburg.de).
spatial directions during seasonal migration or homing (Wehner and Labhart 2006). Celestial polarized light signals are detected by photoreceptors in a specialized region of the compound eye, the dorsal rim area (DRA) (Labhart and Meyer 1999). While diurnal insects including ants, bees and monarch butterflies refer to polarized light generated by the sun (Frost and Mouritson 2006; Wehner 1984), nocturnal dung beetles rely on the dim polarization pattern produced around the moon (Dacke et al. 2003; 2004).
Desert locusts (Schistocerca gregaria) perform long-distance migrations in huge swarms throughout North Africa and the Middle East and have been used as model organisms to analyze neural networks underlying the processing of sky compass signals in the brain. Behavioral experiments on tethered flying locusts suggest that they are able to use polarized light signals from the blue sky to define their course during migration (Mappes and Homberg 2004). Like other locust species, desert locusts occur in two phases, a gregarious and a solitarious phase, that show substantial differences in appearance and behavior (Uvarov 1966, Simpson et al. 1999). While gregarious locusts migrate in swarms during the day, solitarious locusts are nocturnal and preferentially migrate as individuals during the night (Waloff 1963; Roffey 1963). Considerable differences in the size and proportion of brain areas underlying the processing of visual signals were found between both phases (Ott and Rogers 2010), and, at the neural level, differences were demonstrated in the physiology of a looming-sensitive interneuron (Rogers et al. 2010).
Polarized light information is processed in distinct areas in the locust brain (Homberg 2004;
Homberg et al. 2011). The anterior optic tubercle (AOTu) is a major relay station for processing polarized light information and transfers polarized light signals from the optic lobe to the central complex (Homberg et al. 2003). The tubercle receives signals from the dorsal rim area of the medulla and layer 4 of the distal medulla via transmedulla neurons (el Jundi et al. submitted). Two classes of polarization-sensitive (POL)-neurons, intertubercle cells and neurons of the tubercle-lateral accessory lobe tract, were identified in the AOTu (Pfeiffer et al.
2005). Three intertubercle neurons, a single LoTu1 and a pair of TuTu1 cells interconnect the AOTu of both hemispheres (Pfeiffer et al. 2005). The second class of neuron, TuLAL1-cells, transfers polarization signals to input neurons of the central complex (Träger et al. 2008). POL-neurons typically are
POL-neurons of the AOTu in gregarious and solitarious locusts
47
with a rotating polarizer (Labhart 1988). Except for the LoTu1 cell, all POL-neurons of the AOTu show polarization opponency, i.e. they are maximally activated at a distinctE-vector orientation (fmax) and are maximally inhibited at an orthogonal orientation (fmin) (Pfeiffer et al. 2005). The LoTu1 neuron lacks an inhibitory part at fmin, suggesting a particular role in the neural network of the AOTu.
In addition to polarized light, all POL-neurons of the AOTu also respond to unpolarized chromatic stimuli which might allow them to distinguish between the solar and antisolar hemispheres of the sky (Kinoshita et al. 2007; Pfeiffer and Homberg 2007).
Orientational tuning of these neurons to polarized light signals varies in a daytime dependent manner suggesting that the neurons compensate for daytime changes in solar elevation (Pfeiffer and Homberg 2007; Homberg et al. 2011).
To further characterize the signaling properties of the AOTu neurons, we have studied their receptive field properties, intensity/response functions, and daytime dependent differences in sensitivity to polarized light. To reveal possible adaptations to different lifestyles, data were obtained from gregarious and solitarious locusts. Surprisingly, we found no differences between solitarious and gregarious locusts in the physiological parameters of these neurons. In contrast, the properties of the different types of AOTu neurons differed markedly.
TuTu1 and TuLAL1 neurons appeared to be adapted to signal E-vector orientation during the day independent of light intensity, while LoTu1 showed an increased sensitivity and response amplitude during the night suggesting optimal signaling of E-vector contrast under twilight conditions. Therefore, gregarious and solitarious locusts might possess similar adaptations for high and low light intensity detection of the sky polarization pattern.
M E T H O D S
Locust rearing
Gregarious desert locusts (Schistocerca gregaria) were raised under crowded conditions at a constant temperature of 28°C on a 12h:12h light/dark cycle. Rearing conditions for solitarious animals followed the procedures of Roessingh et al. (1993). Animals were kept individually in small boxes at 26.5°C, 60% humidity and 12 h light/dark photoperiod and had neither visual nor olfactory contact. In general, full transition to the solitarious phase required three generations of animals kept in isolated conditions. A number of morphological markers were used as indicators for successful generation of solitarious animals. Solitarious nymphs had a bright green coloration in contrast to a yellow-dark brown patterning of gregarious nymphs (Simpson et al. 1999). Freshly hatched adults were light green in the solitarious state but had a pinkish coloration when they were gregarious. Sexually mature males were of yellow color with black patches in the gregarious state and were more uniformly brown-grey colored as solitarious animals. Another marker for solitarious adults was a light
midline stripe along the dorsal thorax, which was less prominent in gregarious animals.
Preparation and electrophysiology
Only sexually mature locusts (1-3 weeks after imaginal molt) were used for the experiments.
Recordings were performed from AOTu neurons during the subjective night and subjective day of the animals. In both cases preparation of the animals was performed under identical conditions using a cold light source (Leica, KL 1500, Leica Microsystems, Wetzlar, Germany) for illumination.
Animals were cold anesthetized for at least 30 min.
Legs and wings were cropped and stumps were closed with glue or wax. Mouthparts were sealed with wax, and animals were mounted with tape to a metal holder in a vertical orientation. A ridge of wax was brought up frontally between the mouthparts and the anterior edge of the compound eyes. The head capsule was opened anteriorly, and fat and trachea surrounding the brain were removed. To obtain stable recordings, the esophagus was cut, the abdomen was opened posteriorly, and the gut was removed from the opened abdomen. The abdomen was sealed with a tightly knotted thread. A wire platform was inserted between the esophageal connectives and was fixed at the ridge of wax to increase stabilization. Electrode penetration was facilitated by removing the neural sheath at the right anterior optic tubercle. During the whole preparation procedure, lasting for about 45 minutes and during recording of neurons, the brain was immersed in locust saline (Clements and May 1974).
Neurons of the AOTu were recorded intracellularly using sharp microelectrodes (resistance: 60-190 O +0"The electrodes were drawn from borosilicate capillaries (inner diameter: 0.75 mm; outer diameter: 1.5 mm; Hilgenberg, Malsfeld, Germany) using a Flaming/Brown horizontal puller (P-97, Sutter, Novata, CA). Tips of the glass micropipettes were filled with 4% Neurobiotin (Vector Laboratories, Burlingame, UK) in 1 M KCl and shanks, with 1 M KCl. A silver wire inserted into the hemolymph solution served as reference electrode. Neural activity of neurons of the AOTu was amplified (10×) with a custom-made amplifier and monitored with an oscilloscope (Hameg HM 205–3, Frankfurt/Main, Germany). After digitizing at a sampling rate of 5 kHz (CED 1401 plus, Cambridge Electronic Design, UK), signals were stored on a personal computer using Spike2 software (version 6.02; Cambridge Electronic Design, UK). After recording, a constant depolarizing current was used to inject Neurobiotin iontophoretically into the neurons (2-3 nA, 1-5 min).
Stimulation
Locusts of both phases were stimulated with polarized monochromatic blue light obtained from a xenon lamp (XBO 150W, LOT-Oriel Group; Darmstadt, Germany, photon flux 1.8 ×1013 photons/cm2s, 30.82 µW/cm2), after passing a monochromatic filter (450 nm), a light guide (Schölly Fiberoptic, Denzingen, Germany) and a motor-driven linear polarizer (HNP’B, Polaroid, Cambridge, MA).
The polarization filter was rotated through 360° in clockwise (0-360°) and counter clockwise (360-0°) directions with a constant speed of 30°/s. A set of neutral density filters between the light guide and the xenon lamp allowed changing the light intensity in logarithmic steps.
POL-neurons of the AOTu in gregarious and solitarious locusts
The polarization filter and the end of the light guide were attached to a perimeter device that enabled to test the neuronal responses to stimulation from various points along the left-right meridian. In one experiment, ocular dominance was tested by shielding one eye from the light source with a handheld piece of cardboard during stimulation with zenithal polarized light. Recordings were performed under dim ambient light conditions. During intensity/response measurements background light was reduced further by covering the front of the Faraday cage with a light-tight curtain.
Zenithal stimulation of the animal was defined as 90°
elevation, lateral stimulations at an angular distance of 90°
from the zenith were defined as 0° ipsilateral or contralateral stimulation. The terms ipsi- and contralateral refer to the position of the soma of the recorded neuron. The angular size of the stimulus at the locust eye was about 4.7°.
For stimulation with zenithal polarized bright white light (39.17 mW/cm2) the 450nm-monochromatic filter was moved out of the light beam.
Histology
To determine the morphology of the recorded neuron, Neurobiotin was injected into the recorded neuron. Brains were dissected out of the head and were fixed over night in 4% paraformaldehyde at 4 °C. Then, brains were washed 4
× 15 min with 0.1 M phosphate buffered saline (PBS, pH 7.4) and were incubated with streptavidin conjugated to Cy3 (1:1000; Dianova, Hamburg, Germany) in 0.1 M PBS containing 0.3% Triton X-100 (PBT). After an incubation period of three days, brains were again rinsed two times in 0.1 M PBT and then in 0.1 M PBS and were dehydrated in an ascending ethanol series (25%-100%, 15 min each).
After treatment with a solution of ethanol/methyl salicylate (1:1, 15 min), brains were cleared in methyl salicylate for at least 35 min. The wholemount preparations were finally embedded in Permount (Fisher Scientific, Pittsburgh, PA, USA) between two glass coverslips using ten reinforcement rings as spacers (Zweckform, Oberlaindern, Germany).
Data analysis
The sampled spike trains were evaluated by using the Spike2-software with a custom designed script (kindly provided by Dr. K. Pfeiffer, Halifax, Canada). Action potentials were detected through threshold-based event detection. Events were visualized as mean spiking frequency using a gliding average algorithm (moving average of firing rate in window size: 1s). Background activities of the recorded cells were measured by counting of spikes divided by the respective time in a part of the spike train without stimulation. To define the E-vector tuning of the neurons, events during clockwise and counter clockwise rotations of the polarizer were assigned to the corresponding E-vectors and lists of these angles were analyzed using Oriana 2.02 software (Kovach Computing Services, Anglesey, UK). The angle of the mean vector r averaged from equal numbers of clockwise and counter clockwise rotations of the polarizer was defined as the E-vector tuning (fmax) of that neuron. In addition, the length of r describes the concentration of action potentials around fmax and is, thus, a measure for the directedness of the response during rotation of the polarizer (Batschelet 1981;
Pfeiffer et al. 2011).
To quantify the modulation strength of the neurons
response strength R (Labhart 1996). The stimulation period of the rotating polarizer was divided into 18 consecutive bins of 20°. In each bin we calculated the difference between the actual spike frequency and the mean spike frequency during the total stimulation period. The sum of the absolute value of all 18 bins was defined as the response strength R. Background variabilities of the cells were calculated in the same way in a section of the spike train without stimulation. Relative R values were obtained by normalizing the modulation strength at a given position of the visual field to the maximum value (Rnorm). The widths of the receptive fields were determined by analyzing the elevations of half-maximal response strength in relation to the background variability. For visualization, data points of the receptive fields were connected by lines." fmax -distributions within the receptive field were obtained by subtracting"vjg"cduqnwvg"fgxkcvkqp"qh"vjg"fmax value at each elevation from the preferred zenithal orientation.
In intensity/response diagrams, the response strengths were normalized against the modulation strength at log 0 (Rnorm). Intensity/response curves were fitted by applying a modified Naka-Rushton function to the data (Naka and Rushton 1966)
)
(max)
(
n nn
K I R I
R
norm norm× +
=
(1)where I is the intensity of the stimulus, K is the intensity of the stimulus at 50% Rnorm(max), and is an exponent.
Box plots were created with the software Origin 6.0 (Microcal, Northhamton, CA, USA). The median value was indicated through a horizontal line and boxes denoted the 25% and 75% quartiles of the data. The 5% and 95% range of the data were visualized through whiskers.
Statistics
Circular statistics were performed in Oriana 2.02.
Responses of neurons to polarized light were analyzed statistically through the Rayleigh test for axial data (Batschelet 1981). Neurons were defined as polarization sensitive if the distribution of angles was significantly fkhhgtgpv"htqo"tcpfqopguu"*g?2027+0 The distribution of the preferred orientations of different recordings from the same neuron type was analyzed through Rao’s spacing test (significance level, 0.05)." Vq" vguv" yjgvjgt" vjg" fmax -distribution of corresponding neurons differed between solitarious and gregarious animals, the Watson-Williams F test (significance level, 0.05) was used.
Further quantitative comparisons of the data were made by using the SPSS software (Version 11.5). The Shapiro-Wilk test (significance level, 0.05) was used to test for normality of data and the Levene test (significance level, 0.05) to test for homogeneity of variance. For data that were not distributed normally or if the variance was inhomogeneous the Mann-Whitney U test (significance level, 0.05) was applied. In the case of a normal distribution of the data and homogeneity of variance, the two samples were analyzed through a student’s t test (significance level, 0.05). If data were compared from the same recorded neuron, quantitative analysis was performed through a paired student t test (significance level, 0.05). For statistical evaluation of multiple groups a one-way ANOVA combined with Tukey-honestly significant difference (HSD) post hoc test was applied (significance level, 0.05). If the
Shapiro-POL-neurons of the AOTu in gregarious and solitarious locusts
49
Games-Howell post hoc test was used (significance level, 0.05). Linear regressions were calculated using Origin 6.0.
The correlation coefficient (Rcorr) was measured and the significance of regression was tested through a t test against a slope of 0 (significance level, 0.05).
R E S U L T S
This study presents electrophysiological data from 113 intracellular recordings from polarization-sensitive (POL) neurons of the anterior optic tubercle (AOTu) in the locust brain. Four types of neuron were analyzed, two types of intertubercle neuron that transfer polarization information from the ipsilateral to the contralateral AOTu and two types of neuron that connect the AOTu to the lateral accessory lobe.
The lobula-tubercle neuron 1 (LoTu1) exists as a single neuron per hemisphere. It arborizes in the lower units of both AOTus and has further ramifications in the ipsi- and contralateral anterior lobulae (Vitzthum et al. 2002). In contrast, the arborizations of the tubercle-tubercle neuron 1 (TuTu1, 2 neurons per hemisphere) are restricted to the lower units of the AOTus of both hemispheres.
The other two types of neuron, termed tubercle-lateral accessory lobe neurons (TuLAL) consist of about 100 neurons per brain hemisphere (Homberg et al. 2003).
TuLAL1a neurons connect the AOTu with the ipsilateral lateral accessory lobe via the tubercle-accessory lobe tract (Pfeiffer et al. 2005). TuLAL1b neurons ramify in the anterior lobula, the AOTu and the lateral accessory lobe (Pfeiffer et al. 2005).
Receptive field structure and general tuning of AOTu neurons in gregarious and solitarious locusts
TuTu1 intertubercle neurons were analyzed in 20 recordings from gregarious animals and 13 recordings from solitarious locusts (Fig. 1). TuTu1 neurons responded with polarization opponency to a dorsally rotating polarizer with excitation at fmax and inhibition at fmin (Figs. 1A,B). TuTu1 neurons from gregarious locusts had a background activity of 25.5 ± 11.4 (SD) imp/s and a background variability of 38.9
± 15.0 (SD). They showed an average absolute response strength R of 176.2 ± 98.9 (SD) to polarized light stimulation. The fmax values of the gregarious TuTu1 neurons had three preferred orientations, one at approximately 36°, another around 120° and a third at around 175°. Receptive field properties of TuTu1 neurons of gregarious animals were analyzed in 19 recordings. In 16 recordings the bilateral expansion of the receptive fields was analyzed during the subjective day (Zeitgeber time 0h-12h), while in three gregarious locusts receptive field properties were analyzed between Zeitgeber time 12 to 24h (subjective night). No differences between the receptive fields of TuTu1 cells recorded at night or during the day were noted. The averaged receptive field of all 19 TuTu1 neurons had a width of about
110° and was centered eccentrically at an elevation of 60° in the contralateral hemisphere (Fig. 1C).
TuTu1 neurons of solitarious animals had a mean background activity of 29.1 ± 10.87 (SD) imp/s and a mean background variability of 44.9 ± 23.52. Both values did not differ significantly between solitarious and gregarious animals (student’sttest, p = 0.38, p = 0.33, respectively). TuTu1 neurons from solitarious locusts had an averaged response strength R of 144.20
± 61 (SD), which was not significantly different from that of TuTu1 cells from gregarious animals (Mann-WhitneyU test, p = 0.55). fmax orientations of TuTu1 neurons were distributed more randomly in solitarious locusts (Fig. 1E), but this distribution did not differ significantly from the distribution offmaxorientations of TuTu1 cells in gregarious locusts (Watson-Williams F test, p = 0.8). Receptive field properties were analyzed in 11 neurons at night and 2 neurons during the day, but as in gregarious animals no differences were observed between the two groups.
The averaged receptive field of all TuTu1 cells of solitarious animals had a width of about 120°. Similar to the receptive field in gregarious locusts, it was centered eccentrically between 60° and 30° in the contralateral hemisphere (Fig. 1C). No significant differences were observed between solitarious and gregarious locusts at any tested position in the visual field.
The LoTu1 neuron was analyzed in 69 experiments (Fig. 2). In contrast to TuTu1 neurons, LoTu1 was most strongly activated at fmaxbut lacked an inhibition at fmin (Figs. 2A,B). In 40 recordings, LoTu1 properties were tested in gregarious animals.
The neurons had a background activity of 13.3 ± 10.1 (SD) imp/s, a mean background variability of 21.3 ± 7.3 (SD), and a response strength R of 72.4 ± 27.8 (SD) in the center of the receptive field. The fmax
orientations of the recorded neurons in gregarious animals showed a non-random distribution (Rao’s spacing U test, p < 0.01) and ranged – with three exceptions – from about 76° to 176°, with a mean fmax orientation at 128.4° ± 31.6 (SD) (Fig. 2D). The receptive field structure of LoTu1 was analyzed in 33 gregarious animals. 26 recordings were obtained during the subjective day and in seven animals recordings were performed during the night. No significant differences were found between receptive fields of both groups at any of the tested elevations (ANOVA analysis with Games-Howell post hoc test).
Similar to TuTu1 cells, the gregarious LoTu1 neuron had an eccentric receptive field with the strongest response at an elevation of 60° contralaterally (Fig.
2C). The width of the receptive field was about 130°
along the left-right meridian.
Physiological properties of the LoTu1 cell in solitarious locusts were analyzed in 26 animals. The averaged background activity of 14.9 ± 8.1 (SD) imp/s and the mean background variability of 19.4 ± 6.1 (SD) was not different from the corresponding firing properties in gregarious animals (Mann-Whitney U test, p=0.34, p = 0.25, respectively). The neurons showed an absolute response strength of 85.9
POL-neurons of the AOTu in gregarious and solitarious locusts
FIG. 1. Physiology of TuTu1 neurons from gregarious and solitarious locusts. A: Spike train of a TuTu1 neuron from a gregarious animal during dorsal stimulation with polarized blue light. The polarizer was rotated in clockwise direction; lower trace: spike train; upper trace:
mean spiking frequency (moving average of spike rate in 1s time window). B: Circular plot of the mean spiking rate of the neuron shown in A plotted against the E-vector orientation of the polarizer (bin size: 10°; n=4; error bars = standard deviation, fmax = 174°, Rayleigh test, p <
10-12). Grey circle indicates the background activity of the neuron in darkness. C: Mean response amplitudes of TuTu1 neurons from gregarious (n = 19, black) and solitarious (n = 13, grey) locusts along the left-right meridian. Normalized response strength (Rnorm) was measured at different elevations in the ipsilateral (i) and contralateral (c) field of view. For better visualization, data points are connected by solid lines. Response amplitudes and mean background variabilites (dotted lines) were normalized to the maximum R value in the visual field of each neuron. In gregarious animals, eight neurons showed the strongest response at an elevation of 30° contralaterally (30c), seven cells at an elevation of 60° contralaterally (60c), one neuron at zenithal stimulation (90), and three neurons in the ipsilateral visual field (60i).
Of a total of 13 receptive fields analyzed in solitarious animals, five neurons showed the strongest modulation at an elevation of 30°
contralaterally (30c), four neurons at 60° contralaterally (60c), and three neurons at zenithal stimulation (90). Error bars denote standard errors. D: Distribution of fmax orientations of TuTu1 neurons of gregarious locusts obtained during zenithal stimulation (n = 16; bin width:
10°). E: Distribution of zenithal E-vector orientations of TuTu1 neurons from solitarious animals plotted against the number of recorded neurons (n = 13; bin size: 10°). All values are plotted for cells with perikarya in the left brain hemisphere. fmax values of neurons with cell bodies in the right hemisphere were mirrored against the longitudinal axis of the animal.
± 33.16 (SD) in the center of the receptive field which did not differ significantly from the response strength in gregarious locusts (student t test, p = 0.1). fmax
orientations of LoTu1 neurons in solitarious animals were distributed more randomly (Figure 2E), but statistically no differences were observed between the E-vector tuning in gregarious and solitarious animals (Watson-Williams Ftest, p=0.07). The receptive field properties of LoTu1 neurons from solitarious animals were studied in 25 recordings (nine cells at the subjective day and 16 neurons at Zeitgeber time 12-24h). Again, no significant differences were found in the receptive field properties between gregarious and solitarious locusts that were recorded during the subjective night or the subjective day (ANOVA analysis with Games-Howell post hoc test). In all groups, LoTu1 neurons had a receptive field of highly similar width (about 135°) and shape (Fig. 2C). As in gregarious locusts, the strongest response of LoTu1 in
solitarious locusts was centered at an elevation between the zenith and 60° contralateral.
Owing to the small diameter of TuLAL1 neurites, recordings from these neurons were relatively difficult and, thus, in previous work these types of neuron were analyzed only rarely. We studied TuLAL1 neurons in eleven recordings (Figs. 3,4). The size of the receptive field along the left-right meridian of TuLAL1a cells was analyzed in seven recordings (two gregarious and five solitarious locusts). In all recordings, TuLAL1a neurons showed polarization opponency (Figs. 3A,B). The background activity of the two gregarious TuLAL1a neurons ranged from 36.2 to 45.5 imp/s and the background variability ranged from 16.5 to 34. Both receptive fields were zenith-centered and quite narrow (about 60°) (Fig.
3C). The response strength R of both cells ranged from 105.49 to 115.74. The fmax orientation of both neurons was around 30° whereas the fmax orientations
POL-neurons of the AOTu in gregarious and solitarious locusts
51
FIG. 2. Analysis of LoTu1 neurons in gregarious and solitarious locusts. A: Unfiltered spike train (lower trace) and mean firing frequency (upper trace) of a LoTu1 neuron during stimulation with polarized blue light (clockwise rotation) obtained from a gregarious animal (moving average, bin size: 1s). B: Circular diagram of the mean spike frequency of the neuron shown in A plotted against the orientation of the polarizer (bin size: 10°; n = 6; error bars = standard deviation, fmax = 94°, Rayleigh test, p = 5.74 × 10-6). Grey circle shows the background activity of the LoTu1 neuron. C: Averaged receptive field width along the left-right meridian of LoTu1 analyzed in gregarious (n = 33, black) and solitarious (n = 25, grey) animals. Relative response strength (Rnorm) is plotted at different elevations of the polarizer along the right left meridian of the visual field. In each neuron, R and the mean background variability (dotted lines) were normalized to the highest R value in the visual field. In gregarious locusts, one neuron responded maximally to polarized light from the contralateral horizon (0c), two neurons at an elevation of 30° contralaterally (30c), and 16 neurons at an elevation of 60° contralaterally (60c). Ten neurons showed the strongest sinusoidal modulation during presentation of polarized light from dorsal (90), two neurons at 60° ipsilaterally (60i), and two further cells at an elevation of 30° in the ipsilateral hemisphere (30i). In solitarious animals, eight neurons responded maximally to polarized light at an elevation of 30° in the contralateral field of view (30c), six neurons at a position of 60° in the contralateral hemisphere (60c), and eight cells during zenithal stimulation with polarized light (90). Three LoTu1 neurons from solitarious animals showed the strongest responses in the ipsilateral hemisphere (two neurons at 60i, one cell at 30i). Error bars show standard error. D: fmax distribution of LoTu1 neurons (n = 29) from gregarious animals. Only neurons that showed significant responses during zenithal stimulation were considered. The distribution of the preferred orientations to polarized light differed significantly from a uniform distribution (mean fmax angle: 131° ± 29.4° (SD); Rao’s spacing test, p< 0.01, bin size: 10°). E: The distribution of fmax from solitarious animals (n = 24) did not differ from randomness (Rao’s spacing test, p> 0.05; bin width: 10°). All values were treated as if originating from neurons with somata in the left brain hemisphere. For cells with cell bodies in the right hemisphere, values were mirrored against the longitudinal axis of the animal.
of the five TuLAL1a neurons from solitarious animals were distributed randomly (Fig. 3F). Without stimulation, neurons in solitarious animals had a mean background activity of 38.7 ± 19.66 (SD) imp/s and a mean background variability of 38.5 ± 9.2 (SD). No significant differences were observed in response strength, background activity, and background variability of TuLAL1a neurons between solitarious and gregarious locusts. The receptive fields of the solitarious TuLAL1a neurons varied considerably in bilateral size and position and had centers in the contralateral or ipsilateral hemisphere (Fig. 3D). In one TuLAL1a cell from a gregarious locust, ocular dominance was tested by monocular stimulation of the ipsi- and contralateral eye (Fig. 3E). In contrast to the intertubercle neurons (Pfeiffer et al. 2005), the neuron responded with similar response strength to
ipsilateral, contralateral, and bilateral polarized-light stimulation (Fig. 3E).
Recordings from TuLAL1b neurons were obtained from four gregarious animals (Fig. 4). Three of the four neurons showed polarization opponency (Figs. 4A,B), while one TuLAL1b neuron was only activated during stimulation with polarized light. All four cells arborized in the lateral triangle as well as in the median olive of the lateral accessory lobe. The four neurons had a background activity of 18.5 ± 5.6 (SD) imp/s and a background variability of 34.24 ± 16.9 (SD) in darkness. TuLAL1b neurons had a mean response strength of about 155 ± 46.8 (SD) in the center of the receptive field. As in TuLAL1a neurons, receptive field structures of individual TuLAL1b cells varied substantially in bilateral extension and position of the receptive field along the left-right meridian.
The cells had receptive field centers in the zenith, the
POL-neurons of the AOTu in gregarious and solitarious locusts
FIG. 3. Polarization-sensitive TuLAL1a neurons recorded in gregarious and solitarious locusts. A: Neural activity and mean firing rate of a TuLAL1a neuron during zenithal stimulation with a rotating polarizer (clockwise rotation, blue light, 450nm); lower trace shows the spike train, whereas the mean spiking activity is visualized in the upper trace with a moving average bin size of 1s. B: Circular diagram of mean frequencies of action potentials of the neuron in A plotted against E-vector orientation of the polarizer (n = 4, error bars = SD, bin size: 10°;
fmax = 158°; Rayleigh test, p < 10-12). Grey circle indicates background firing activity without stimulation. C, D: The normalized modulation strength (Rnorm) of two gregarious (C) and five solitarious (D) animals plotted against the elevation of the stimulus along the left-right meridian. In each neuron, the R value was measured at different elevations and was normalized to the strongest response of the neuron in the visual field. Normalized variabilities of firing activity in darkness are shown as dotted lines. E: Ocular dominance test of a TuLAL1a neuron from a gregarious locust. Grey bar shows the background variability of the neuron. The response strength R for zenithal monocular stimulation of the ipsilateral eye (ipsi) and contralateral eye (contra) with a rotating polarizer was normalized to the response strength (dotted line) for binocular stimulation. Error bars = SD. F: The distribution of"fmax orientation analyzed in the center of the receptive fields of seven TuLAL1a neurons recorded from gregarious (black bars, n = 2) and solitarious (grey bars, n = 5) animals plotted against the number of recorded neurons (bin size: 10°). All values are plotted as if originating from neurons with somata in the left brain hemisphere.
The cells had receptive field centers in the zenith, the ipsilateral or the contralateral hemisphere. Preferred E-vector orientations in the receptive field center were between 130° to 180° in three neurons and about 5° in one neuron.
Taken together, no differences in the general physiological properties and in receptive field structures of POL-neurons of the AOTu between gregarious and solitarious locusts were observed.
Both intertubercle neurons had large receptive fields centered to the contralateral hemisphere. In contrast, the receptive fields of TuLAL1 neurons were considerably more narrow and varied substantially in shape and position.
Intensity-response functions of AOTu neurons in solitarious and gregarious locusts
While gregarious locusts migrate during the day, solitarious animals preferentially migrate during the night (Walloff 1963; Roffey 1963). We were therefore interested to see whether these different lifestyles are reflected in the polarization vision network in the locust AOTu. Intensity/response (I/R) functions were obtained by changing the intensity of the polarized
over a range of 4 log units (Fig. 5). Neurons recorded during the subjective day (Zeitgeber time 0h-12h) were treated separately from neurons recorded during the subjective night (Zeitgeber time 12h-24h).
TuTu1 neurons were analyzed during the subjective day in 7 gregarious and 6 solitarious animals (Fig. 5A). The response strengths of the gregarious TuTu1 neurons were saturated between log I = 0 and log I = -2 and showed a sharp drop to background levels between log I = -3 and -4 (Fig. 5A).
The I/R function of solitarious animals was intensity-independent between log I = 0 and -3, but at a logarithmic step of -4 the response broke down to background levels (Fig. 5A). Statistically, no differences were observed at each intensity step of the I/R function of TuTu1 neurons between gregarious and solitarious locusts.
I/R functions of LoTu1 neurons are based on 24 recordings in gregarious locusts and 18 recordings from solitarious animals. 18 LoTu1 neurons of gregarious locusts and five LoTu1 neurons of solitarious animals that were recorded during the day (Fig. 5B) showed similar I/R curves that gradually decreased to background levels between log I = 0 and log I = -4. In addition, recordings from seven LoTu1 neurons in gregarious animals and 13 LoTu1 neurons
POL-neurons of the AOTu in gregarious and solitarious locusts
53
FIG. 4. Physiological analysis of TuLAL1b neurons from gregarious animals. A: Spike train (upper trace) and mean spiking frequency (lower trace) of a TuLAL1b neuron during zenithal stimulation with a polarizer that rotated in counter clockwise direction (moving average, bin width: 1s). B: Circular plot of the mean firing rate of the TuLAL1b neuron in A plotted against the orientation of the polarized light-stimulus (n = 6."gttqt"dctu"?"UF."dkp"uk¦g<"32̇="fmax = 7°; Rayleigh test, p = 1.22 × 10-5). Grey solid circle shows the background activity of the neuron. C: Receptive field properties along the left-right meridian of four neurons from gregarious locusts. For each neuron, the modulation strength R was measured at different elevations of the ipsilateral (i) and contralateral (c) field of view and was normalized to the maximum R value in the receptive field (Rnorm). Mean background variability is denoted as dotted line. D: The distribution of preferred E-vector orientations of the four TuLAL1b neurons in the center of the receptive fields. Data are plotted as if originating from neurons with perikarya in the left brain hemisphere of the animal.
similar sensitivity curves (Fig. 5C). As in TuTu1 neurons, I/R curves from LoTu1 did not differ significantly between solitarious and gregarious locusts.
As mentioned earlier, the response value R is a measure for the modulation strength of firing activity during stimulation but does not give information about the directedness of the response. Therefore we tested whether the length of the mean vector r,which serves as a measure for the directedness of the response to polarized light (Pfeiffer et al. 2011), differed between both locust phases (Fig. 5C,D). No significant differences in the directedness of TuTu1 (Fig. 5C) and LoTu1 neurons (Fig. 5D) between solitarious and gregarious locusts were found. Taken together the data suggest that there are no differences in the neural network of the AOTu underlying the processing of polarized light between both locust phases.
Differences in neural responses between AOTu neurons
The I/R curves between both types of intertubercle neurons differed substantially. Whereas the response strength to polarized light of the TuTu1
neurons remained relatively constant between log I = 0 and log I = -3 but declined to background levels within the final log unit (Fig. 6A), the modulation strength in the LoTu1 neuron decreased gradually from one logarithmic intensity step to the next (Fig.
6B). This is also reflected in the statistical analysis: In TuTu1 neurons the response at log I = 0 differed only from the response at the lowest light intensity step (log I = -4, Fig. 6A), whereas in the LoTu1 neuron the response to the highest analyzed light intensity differed significantly from all other light intensities (Fig. 6B). Furthermore several light intensity steps in LoTu1 differed significantly among each other. The I/R curves of the TuLAL1a and TuLAL1b neurons were similar to the I/R function of TuTu1 neurons, but showed a slightly more shallow decline between log I
= -2 and log I = -4 to baseline levels (Figs. 6C,D).
Thus, in contrast to TuTu1 and TuLAL1 neurons which may signal E-vector orientation above threshold levels independent from light intensity, the response in LoTu1 is strongly dependent on the intensity of the polarized light throughout all intensities tested.
We next compared the response properties of the neurons in greater detail to further characterize the distinct roles of the different cell types in the processing of polarized light. Because no differences
POL-neurons of the AOTu in gregarious and solitarious locusts
FIG. 5. Normalized intensity/response (I/R) functions of intertubercle neurons of gregarious and solitarious locusts to stimulation with polarized blue light in the center of their receptive fields. Maximum light intensity (log I = 0) was 1.8 ×1013 photons/cm2s. The response strength R (A,B) and the length of mean vector r (C,D) were calculated at each light intensity. Solid curves in A and B are fitted through a modified Naka-Rushton function, and dotted lines denote the background variability. In C,D the data points are connected through solid lines for better visibility; broken lines show the directedness of the cells without stimulation. Error bars in all diagrams indicate standard errors. A:
No differences are observed in the I/R function of TuTu1 neurons from gregarious locusts (n = 7, black fit, Naka-Rushton fitting parameters, Rnorm(max) = 1.02, K = -3.5 log units, "?"2096+"cpcn{¦gf"fwtkpi"vjg"fc{"*Zeitgeber Time (ZT): 0-12h) and solitarious animals (n = 6, grey curve, Rnorm(max)= 1.01, K = -6056"nqi"wpkvu." "?"305+"tgeqtfgf"fwtkpi"vjg"pkijv"*¥V<34-24h) (log I = -1, -3 and -4 are tested through a student t test; log I = -2 tested through a Mann-Whitney U test, p > 0.05). B: I/R functions of LoTu1. Left figure shows the I/R functions from gregarious locusts (n = 18, black line, Rnorm(max) = 1.12, K = -408"nqi"wpkvu." "?"204;+"cpf"uqnkvctkqwu"cpkocnu"*p"?"7."itg{"nkpg. Rnorm(max) = 1.27, K = -0.19 log units,
"?"2048+"cpcn{¦gf"fwtkpi"vjg"fc{0"Vjg"tkijv"fkcitco"ujqyu"K1T"hwpevkqpu"qh"NqVw3"pgwtqpu"htqo"itgictkqwu"*p"?"9."dncem"rnqv."Tnorm(max) = 1.05, K = -30;7"nqi"wpkvu." "?"204;+"cpf"uqnkvctkqwu"cpkocnu"*p"?"35."itg{"hit, Rnorm(max) = 0.92, K = -50;;"nqi"wpkvu." "?"208;+""ogcuwtgf"cv"¥V"34"
-24. No differences were found between gregarious and solitarious LoTu1 neurons and between the I/R curves at ZT 0-12 and ZT 12-24 (log I
= -1 and log I = -4 tested through an ANOVA analysis with Games-Howell post hoc test, log I = -2/-3 analyzed through an ANOVA combined with Tukey-HSD post hoc test, p > 0.05). C,D: Directedness of the response at different intensities of polarized light; same set of neurons as in A,B. No significant differences are present in TuTu1 neurons (C, log I = -1/-2 tested through a student t test; log I = -3/-4 tested by a Mann-Whitney U test) and in LoTu1 neurons (D, ANOVA analysis with Games-Howell post hoc test) between the two locust phases.
between solitarious and gregarious locusts were found in general tuning characteristics and light intensity dependence, data from both forms were pooled for the following analyses. LoTu1 neurons showed a significantly lower background firing rate than TuTu1 and TuLAL1a neurons (Fig. 7A). Furthermore the background spiking rate in darkness was significantly lower in TuLAL1b cells than in TuLAL1a neurons.
While LoTu1 neurons showed low background variability and response strength to polarized light stimulation, TuTu1 neurons showed significantly higher background firing variability and a higher response strength R (Fig. 7B,C). No statistical differences were observed between TuLAL1a and TuLAL1b neurons in background variability and response strength R and between the intertubercle cells and the TuLAL1 neurons. Finally, the directedness of the response showed no differences between all AOTu neuron types (Fig. 7D).
In the next step we analyzed possible correlations between the tuning characteristics. Not surprisingly, the response strength of all cell types correlated
(data not shown). However, in all other tuning properties, no significant correlations were found except for a correlation between the background activity and the length of mean vector r. While in TuTu1 neurons no correlation between the background spiking activity and the length of the mean vector r was found (Fig. 7E), a linear correlation was present in LoTu1 (Fig. 7F; t test for slope = 0, R: -0.5, p=0.0001). A similar correlation was found, when all TuLAL1 cells were plotted together (Fig. 7G; t test for slope = 0, R: -0.65, p=0.03).
In the next analysis, the distributions" qh" fmax
within the receptive field were investigated (Fig. 8).
In contrast to TuTu1 cells which did not show systematic changes in the preferred E-vector orientation within the receptive field (Fig. 8A), the preferred E-vector angle of LoTu1 increased within the receptive field from ipsi- to contralateral positions (Fig. 8B, t test for slope = 0, R: 0.23, p=0.005).
Likewise, in TuLAL1a neurons deviations from zenithal fmax–values depended on the hemispheric